High resolution X-ray crystallographic methods are unparalleled toward the chemical study of enzyme mechanism and inhibition. We employ unusual diffraction methods, i.e., high pressure and low temperature protocols, toward the study of carbonic anhydrase II in its complexes with biologically-relevant ligand molecules. Carbonic anhydrase II is one of only two diffusion-control enzyme from the human for which all chemical, kinetic, genetic, and X-ray crystallographic studies are performed on the same enzyme from the same source. In our laboratory, carbonic anhydrase II serves a paradigm for these areas of investigation: (1) the understanding and modification of efficient biological catalysis; (2) protein engineering of zinc binding sites; and (3) rational drug design. The biological substrate of carbonic anhydrase II, carbon dioxide, is a gas under physiological conditions. Hence, ours study of the enzyme-substrate complex is challenged by novel technical methods required for X-ray crystallographic investigation. The structure determination of the enzyme- substrate complex is important to us in order to further our structural intuition of efficient biological catalysis. Additionally, the catalytic versatility of the enzyme is impressive and is actually enhanced in certain site-specific mutants. We will study the three-dimensional structures of site-specific mutants of carbonic anhydrase II where each mutant displays kinetically-significant behavior. Additionally, we will study a mutant enzyme where a significant change has been made in the zinc coordination polyhedron (a histidine-to-cysteine mutation). This will be the first structurally-characterized example of a biological His2Cys zinc coordination polyhedron. Studies of human carbonic anhydrase II -inhibitor complexes have direct pharmaceutical importance given that the enzyme is an established drug target for the treatment of glaucoma epilepsy, and acute mountain sickness. Carbonic anhydrase II is one of but a handful of protein drug targets for which the known three-dimensional structure allows for the direct visualization of protein-drug interaction by X-ray crystallographic methods. As such, it provides an excellent paradigm for developing methods of rational drug design. We will study novel, bivalent inhibitors (and their structural precursors) with the ultimate goal of designing femtomolar inhibitors.